Posttranscriptional mRNA regulation in the yeast colony.

<p>Semi-quantitative ePAT and TVN-PAT reactions were utilized to confirm the expression in the outside or inside colony layers, and to identify differences in 3′UTR dynamics. Included are cDNAs generated from the colony subpopulations, the complete colony, day-4 stationary phase liquid cultures and log phase cultures. The ePAT reaction includes the full poly(A)-tail (seen as a smear of PCR amplicons), whereas the TVN-PAT reaction is anchored to the adenylation site with an invariant A-12 poly(A)-tail (usually a tight band). Multiple bands of different sizes indicates alternate polyadenylation site usage. The site of enrichment for each tested mRNA in the arrays is indicated in brackets (in) or (out) after the gene name. All PCRs were 28 cycles, except <i>ATO1</i> (↑ cycles) panel, where the PCR cycle number was increased to 30. The * indicates alternate transcriptional termination in the SUT350 transcript.</p

Phylograms of the consensus BI tree for the matrix of LEE T3SS genes.

<p>All diagrams are presented with long branches truncated (//) and the branch length indicated above. The subtree in the box represents BI branching on a different scale from the larger tree. Taxa are colored based upon the tRNA insertion point with <i>selC</i> in red, <i>pheV</i> in blue, <i>pheU</i> in green, and black for all other insertion points. Clades are indicated by large Roman numerals above calculated level of genetic diversity (θ) for all the members of the clade. The BI tree is midpoint rooted with posterior probability values represented as a percentage at branch points. ML analysis yielded the same branching (although differential branch lengths) and ML percentage bootstrap values are presented in parentheses () when this value differed from the percent posterior probability. Taxa enclosed by braces had identical sequences to the adjacent taxa and were therefore removed from the phylogenetic analysis.</p

List of selected functional categories, genes and non-coding transcripts enriched on the outside of the colony.

<p>The complete list of genes enriched on the outside of the colony is shown in Dataset S1. CUT-cryptic unstable transcript; SUT-stable unannotated transcript.</p

Gene ontology analysis of differential gene expression within the colony.

<p>Only minimally overlapping GO terms are shown. The full GO analysis is presented in Dataset S1.</p

FACS analysis of yeast strains expressing protein-GFP fusions in stationary phase and colony growth.

<p>Shown are FACS plots. The y-axis represents forward scatter (FSC-A), where an increased signal can indicate increased cell size or budding. The x-axis indicates GFP fluorescence (B525-A). Pink sight lines are included to guide the eye to size and fluorescence differences between the wild-type strain (A) and GFP- fusion stains (B–F). Day-4 yeast cells from the indicated strains were harvested either by washing from the surface of YPAD agar plates (A–D and F), or from stationary phase liquid cultures (E).</p

Noncoding transcription in yeast colonies.

<p>The CUTs (cryptic unstable transcripts) and SUTs (stable unannotated transcripts) were mapped to the genome using the tools at <a href="http://steinmetzlab.embl.de/NFRsharing/" target="_blank">http://steinmetzlab.embl.de/NFRsharing/</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046243#pone.0046243-Xu1" target="_blank">[20]</a>. Examples of the location of noncoding transcripts and their neighboring genes are shown (the drawings are not to scale), and their expression in the outside or inside of the colony is indicated.</p

Schematic of the genomic island containing LEE.

<p>The extent of the genomic island is marked by the solid gray line and the bounding genomic genes outside the genomic island are marked in yellow. Genes that are not part of the LEE region are marked in grey. Genes that form the T3SS are shown in green, the <i>pheV</i> tRNA insertion point is marked by a small red arrow with the remainder of the LEE region in blue. Redundant text from the locus tags has been removed to simplify the figure. The full locus tag of those genes designated with a 4 digit number would read SESS1296_0XXXX. The operon structure of the LEE region is indicated by black arrows. Gene functions are abbreviated, putative integrase (INT), putative transcriptional regulator (TR), putative secreted effector (SE). Groups of genes for which homologs co-occur in other organisms are indicated by curly brackets. Possible pseudogenes are marked with (*). G+C content calculated with a 100 bp window is depicted by the graph below the diagram with maximum (upper line = 61%) and minimum (lower line = 24%) values designated. Typical G+C content of 52% for <i>Salmonella</i> genomes is depicted by the dashed red line. Four tandem repeat regions are depicted by small red boxes. A plot of genetic diversity (θ) calculated for each LEE gene using a Watterson estimate is represented as a spot below the relevant gene.</p

Genes in the SESS LEE genomic island.

a<p>Gene names modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone-0041615-t001" target="_blank">Table 1</a> in Pallen et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone.0041615-Pallen1" target="_blank">[11]</a> with alternative names.</p>b<p>Product names derived from annotation of SESS1296 and modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone-0041615-t001" target="_blank">Table 1</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone.0041615-Pallen1" target="_blank">[11]</a>.</p>c<p>Structural components of the T3SS are indicated with (+).</p>d<p>Diversity (θ) for the listed LEE gene from all alignments including the two <i>Salmonella</i> LEE with standard deviations listed in parentheses.</p>e<p>Diversity (θ) for the listed LEE gene from all alignments excluding the two <i>Salmonella</i> LEE with standard deviations listed in parentheses.</p>f<p><i>Salmonella</i> variants of LEE genes with “orf” designations utilize locus tags as gene names.</p

Rooted phylogenies for T3SS genes <i>escV, escN, escF,</i> and <i>escJ</i>. BI trees for the four T3SS genes described in Castillo et al., [<b>12</b>] are depicted with long branches truncated (//) and the total branch length indicated above.

<p>The subtree in the box represents BI branching on a different scale from the larger trees. Taxa are colored by presence in the clades described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone-0041615-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041615#pone-0041615-g005" target="_blank">5</a> to facilitate comparison between trees (clade I – purple, clade II – green, clade III – blue, clade IV – orange, clade V – red). Taxa enclosed by braces had identical sequences to the adjacent taxa and were therefore removed from the phylogenetic analysis. Diversity values (è) below gene names are calculated excluding the outgroup and those calculations also excluding the SESS LEE genes are in parentheses. Numerals at tree junctions are the percentage posterior probability.</p